Proteins are conveniently produced in a variety of procaryotic and eucaryotic recombinant expression systems. These systems, however, often fail to mimic natural production such that the resulting protein lacks the authentic tertiary conformation and post-translational modifications normally present. Furthermore, expression levels are frequently inadequate, particularly in virally-vectored mammalian systems. For example, in lytic systems, expression can be severely limited by lytic functions of the virus. When high expression levels are achieved, problems with cell growth and expansion can be encountered due to the cytotoxicity of the expressed proteins. Non-lytic systems often suffer from low yields, clone instability and cytotoxicity of the final product.
Inducible expression systems have been employed in an effort to overcome some of these problems. However, most of the inducible promoters currently used in such systems are either restricted to a relatively narrow range of host cells, are only partially inducible or are derived from organisms, such as tumor viruses, which are inherently dangerous. Accordingly, an inducible expression system which provides for the large scale synthesis of proteins, without the above-described concomitant problems, would be highly desirable.
One such candidate is a system using a promoter derived from a group of proteins known as the heat shock proteins (hsps). These proteins are ubiquitous, being found in all eucaryotic organisms studied to date, and are inducible by heat stress, as well as a variety of other external agents. Thus, cells respond to these inducers, such as elevated growth temperatures, by synthesizing high levels of hsps and coordinately reducing the rate of synthesis of other cellular proteins.
Hsps are divided into several groups on the basis of size. Of interest is the hsp70 family, so named because these proteins are approximately 70 kDa in mass. The level of synthesis of hsp70 in cells during heat shock appears to be linearly related to their thermotolerance. Li, G. C. (1985) Int. J. Radiat. Oncol. Biol. Phys. 11:165-177. Two human hsp70 proteins have been described-hsp70A (Wu, B., et al. (1985)Mol. Cell. Biol. 5:330-341; Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. USA 82.:6455-6459) and hsp70B (Schiller, P., et al. (1988) J. Mol. Biol. 203:97-105). For a review of hsps, see, e.g., Morimoto et. al., eds., Stress Proteins in Biology and Medicine (1990) Cold Spring Harbor Press; Hightower, L. E. (1991) Cell 66:191-197.; Craig, E. A., and Gross, C. A. (1991) Trends Bioch. Sci. 16:135.
The hsp70 promoter, as well as sequences in the 5'- and 3'-untranslated regions of hsp70 gene transcripts, are responsible for regulating the level of protein and mRNA synthesis in the cell in both the induced and uninduced states (Simcox, A. A., et al. (1985) Mol. Cell. Biol. 5:3397-3402; Theodorakis, N. G., and Morimoto, R. I. (1987) Mol. Cell. Biol. 7:4357-4368; Yost, H. J., et al. (1990) in Stress Proteins in Biology and Medicine, Morimoto et. al., eds., Stress Proteins in Biology and Medicine (1990) Cold Spring Harbor Press, at 379-409). A region known as the heat shock element (HSE), is found within the first 100 bp 5' of the RNA start site of eucaryotic heat shock genes. Sorger, P. K. (1991) Cell 65:363. This region includes the sequence nGAAn, repeated at least two times in head-to-head or tail-to-tail orientation (nGAAnnTTCn or nTTCnnGAAn). Hsp70 genes from different species differ in the number and orientation of HSEs and in the types of other factor-binding sites found upstream. The HSE functions in stress induced promoter activation by binding a positive transactivating factor, the heat shock factor (HSF). The binding constant of this factor to the heat shock element is about a hundred fold higher than that of any other known mammalian transcription factor to its respective binding site, rendering this promoter one of the strongest.
Hsp promoters have been used to express a variety of genes. For example, Dreano, M., et al. (1986) Gene 49:1-8, describe the use of the human hsp70B promoter, as well as a DroSophila hsp70 promoter, to direct the heat regulated synthesis of human growth hormone, chicken lysozyme and a human influenza haemagglutinin.
EPA Publication No. 336,523 (Dreano et al., published 11 Oct. 1989) describes the in vivo expression of human growth hormone using a human hsp70 promoter.
PCT Publication No. WO 87/00861 (Bromley et al., published 12 Feb. 1987) describes the use of human and Drosophila hsp promoters having 5'-untranslated region variants.
EPA Publication No. 118,393 (Bromley et al., published 12 Sep. 1984) and PCT Publication No. WO 87/05935 (Bromley et al., published 8 Oct. 1987) describe the expression of E. coli .beta.-galactosidase and human influenza haemagglutinin, using a Drosophila hsp70 promoter.
However, none of the above-described references pertains to bovine hsp promoters or to the use of these promoters to drive the expression of heterologous proteins in thermotolerant cells. Nor do any of these references describe the use of an hsp70A promoter for recombinant expression.